Currently there are several technologies available to study molecular complexes, e.g. protein complexes, protein-DNA complexes. Co-Immunoprecipitation (Co-IP) uses different capturing antibodies and detecting antibodies to analyse the interacting protein complex. Proximity ligation assay (PLA) or proximity extension assay (PEA) employs the idea that only when two or more affinity probes bind to adjacent or interacting proteins, the attached DNA oligonucleotides conjugated on the antibody are brought into proximity and thus allowing an enzymatic ligation or extension and formation of a new amplifiable reporter DNA molecule. In the chromatin immunoprecipitation sequencing (ChIP-seq), antibodies are used to capture the protein of interest with its associated DNA fragments. Then the DNA fragments are sequenced to reveal where the protein binds on the chromatin.
These methods have provided useful knowledge when studying the biological regulation at molecular level. However all these methods have the limitation of being unable to identify and quantify all the components from each individual complex, specifically to profile the said complexes. For example, Chip-seq is able to reveal where a protein binds to the chromatin, but it cannot reveal whether two or more proteins bind simultaneously to the same region on the chromatin.
For single cell studies, flow cytometer allows the cells flowing through a thin channel and detects the signal from single cells one by one at a speed as high as several thousand cells per second. To analyse several proteins in parallel, different antibodies can be labelled with different fluorophore. However, spectral overlap can arise when many fluorescent signals are detected simultaneously. To avoid this problem, the mass cytometer, by using mass isotopes to label antibodies, can analyse more than 30 proteins with minimal signal overlap. However, the multiplexing capacity is still limited in the flow-based measurements, and they are not yet suitable for nucleic acid analysis. One way to achieve highly multiplexed analysis for single cells is by sorting cells into separated reaction wells, and analyse the components of each single cell separately. For example in single cell RNA-Seq, single cells are sorted into single wells manually or with the help of automation e.g. fluorescence activated cell sorting (FACS), followed by cell lysis, reverse transcription (RT) and sequencing library preparation with sample barcodes. Then the barcoded products of different cells can be pooled together and sequenced by next generation sequencing (NGS). By this procedure, it's possible analyse several hundreds of cells, but it would be still very laborious to sort many single cells, e.g. more than 10 000, and perform the following library preparation individually. Even with automation system, like C1™ Single-Cell Auto Prep System, preparing the sequencing library of many cells is still a difficult task.
WO2012/042374 aims to provide a method for determining the number or concentration of a molecule in a sample, using nucleic acid molecule tags with unique sequences.
Hindson et al, Analytical Chemistry, 2011, 83, 8604-8610 disclose a high throughput droplet digital system for absolute quantitation of DNA copy number.
WO2012/048341 aims to provide methods and compositions for high-throughput, single cell analyses are provided. The methods and compositions can be used for analysis of genomes and transcriptomes, as well as antibody discovery, HLA typing, haplotyping and drug discovery.
Binladen et al. (PLoS ONE 2(2): e197) used conventional PCR with 59-nucleotide tagged primers to generate homologous DNA amplification products from multiple specimens, followed by sequencing through a high-throughput DNA Sequencing System. Each DNA sequence was subsequently traced back to its individual source through 59tag-analysis.
Landegren et al. (J. Mol. Recognit. 2004; 17: 194-197) discuss using a set of ligation-based reagents termed padlock probes and proximity ligation probes to meet challenges relating to specific detection of all the macromolecules that are being identified in the course of genome projects. The probes include elements with affinity for specific nucleic acid and protein molecules, respectively, along with unique identifier DNA sequence elements that encode the identity of the recognized target molecules.